Heart failure (HF) is a major cause of death, yet understanding of the underlying mechanisms is still limited and treatment options are few at best. This proposal focuses on the elastic protein titin, a recently revealed major player in heart failure, and addresses the basic biology of the poorly understood but clinically important A-band segment of the molecule, the mechanistic basis of its contribution to heart failure, and its potential as a therapeutic target. Titin, the largest known protein, comprises the third myofilament of muscle and spans from the Z-disk to the M-band of the sarcomere. Titin?s I-band region is known to function as a complex molecular spring that is a dominant contributor to passive myocardial stiffness. The A-band segment is the least well- studied part of titin and its functions are largely obscure. Yet several recent landmark sequencing studies in large groups of patients revealed that the A-band segment of titin is particularly important in familial dilated cardiomyopathy (DCM), a common type of heart failure with a prevalence of up to 1:250. Many of the DCM mutations in TTN are truncation variants (TTNtv), most of these are found in the A-band segment of titin, with a phenotype that appears to be more severe the closer the mutation occurs to titin?s C-terminus. In Aim 1 we propose to critically examine the biology of the A-band segment of titin with a focus on where most disease- causing mutations are located, titin?s D-zone and C-zone. Mouse models were created for this purpose in which these regions within the A-band segment of titin were targeted. We propose to study in these models the ultrastructure of the A-band region of the sarcomere, transcript and protein expression (titin, cMyBP-C) and posttranslational-modification (PTMs), as well as heart function. Pilot studies with a novel C-zone deletion model reveal deranged thick filament length and the development of DCM, which provides a unique opportunity to study mechanisms of titin-based DCM. Additional models will be investigated in Aim 2 in which disease-causing TTNtvs were introduced in different regions of titin?s A-band segment. These models will be studied at baseline and when stressed; the severity of their phenotype will be studied as a function of the location of the TTNtv. In parallel, protein expression and structural and functional studies will be conducted on biopsies from DCM patients with TTNtvs. Aim 3 will investigate the new concept in the titin field that TTN is a modifier gene in which disease severity can be explained by a combination of mutations in TTN and other genes. This will be studied by crossing the TTNtv models with mice that carry clinically relevant mutations in other sarcomeric genes. Finally, through excision of the mutated titin exons, we will test the therapeutic potential of exon skipping for treating titin-based DCM. With its basic science and translational goals and its in-depth and integrative approach, this application seeks to continue our track record of cutting edge titin research. Powerful techniques and novel mouse models are in place and pilot data are supportive of our guiding hypotheses. The proposed research is likely to enhance insights in the function of titin titin, including its potential as a therapeutic target.